Increasingly demanding biomedical and biotechnological endeavors require sophisticated materials that can respond to multiple environmental cues, interact with biological systems, template compound assemblies, and direct the growth and differentiation of cells and tissues. My long term research goal is to create customized and functional protein-based biomaterials containing genetically encoded non- standard amino acids (nsAAs) to site-specifically introduce diverse chemical functionalities. Towards this research vision, my research objectives are two-fold. First, I will work to expand and accelerate our ability to produce protein polymers containing multiple nsAAs. To this end I will develop a novel evolution platform and extensive mutagenized libraries for components used to incorporate nsAAs. Second, I will utilize this platform and variants isolated to introduce novel functions and properties to protein-based biomaterials with the goal of developing antimicrobial and drug delivery formulations. This proposal is designed leverage Prof. Isaacs' expertise in synthetic biology, Prof. Soll's expertise in systems for nsAA incorporation, Prof. Saltzman's expertise in biomaterial-based drug delivery, and Dr. Amiram's previous research experience in protein-polymer synthesis, design and application. In addition to scientific training several career development activities will be pursued during this award including attending career development courses offered by Yale University in science and communication skills, developing mentoring skills, and obtaining extensive training in responsible conduct of research. In our preliminary data, we detail a novel evolution platform for aminoacyl-tRNA synthetases (AARS) for nsAA incorporation. We establish that using evolved AARS variants expressed in a genomically recoded E. coli, we are able to bypass former limitations of nsAA incorporation and achieve high yield and high purity production of biopolymers containing multiple (10-30) nsAA instances. In Aim 1, I will extend and expand AARS libraries to enable evolution of translation components for the incorporation adhesive and bulky nsAAs, which can also be expressed and utilized in prokaryotic and eukaryotic organisms. In Aim 2, I will incorporate multiple azide groups to mediate bioorthogonal attachment of multiple fatty acid molecules to improve peptide and protein pharmacokinetics by enhanced albumin binding. In Aim 3, I will generate adhesive DOPA-biomaterials to create silver-containing formulations for antibacterial treatment. Ultimately, I anticipate that my work will facilitate a fundamental change in our abilities to desin and impart new functions and properties to protein- based materials. The results of these studies will yield enabling technologies and strains for multi-site nsAA incorporation, new classes of biopolymers, customized materials with novel functions, and design rules for manipulating and tuning biopolymer properties.